Phytochemical variability between Colombian

accessions of Lippia alba (Mill.) N.E. Brown

Variabilidad fitoquímica entre accesiones
colombianas de Lippia alba (Mill.) N.E. Brown
Variabilidade fitoquímica entre acessos colombianos
de Lippia alba (Mill.) N.E. Brown

José Omar Cardona Montoya1 & Jaime Eduardo Muñoz Flórez 2

Ingeniero Agrónomo, Doctor en Ciencias Agropecuarias. 2Ingeniero Agrónomo,
1

Especialista en Matemática Avanzada, Doctor en Ciencias Agrarias.

1
Docente-investigador Universidad del Pacífico-Buenaventura, Valle. Colombia.
2
Docente-investigador Universidad Nacional de Colombia-Sede Palmira. Colombia.

jocardonam@unipacifico.edu.co, 2jemunozf@unal.edu.co
1

Abstract
The physiological fluctuation of organic constituents
about its environmental response has two compo-
nents, intra-inter individual variation. This study as-
sessed the components of biological variation based
on a matrix of presence-absence, generating preci-
sion values inter-intra individual/population based
on a sample of 58 accessions of L. alba. Chemical
intra-individual variability was high (WI=2.06 [98%])
and the inter-population, was low (AP=0.033), with
FST=0.023 p(rand>data=0.07) and 2% proportion.
There were no high values of Estimates of variance
[Est.Var.] leading to establish chemical variability
among individuals and populations. The results gen-
erated by the test point to the presence of two sub-
populations, defined each by local environmental
conditions.
Resumen
La fluctuación fisiológica de constituyentes orgánicos
alrededor de su respuesta ambiental, tiene dos com-
ponentes, variación intra e interindividual. Este estudio
evaluó los componentes de variación biológica con
base en una matriz de presencia-ausencia, generando
valores de precisión inter-intra poblacional/individual a
partir de una muestra de 58 accesiones de L. alba. La
variabilidad química intra-individual fue alta (WI=2.06
[98%]) y la inter-poblacional, fue baja (AP=0.033), con
FST=0.023 p(rand>data=0.07) y proporción 2%. No
se observaron valores altos de estimativos de vari-
anza [Est.Var.] que conduzcan a establecer diversidad
genética-DG- entre poblaciones e individuos. Los re-
sultados generados por la prueba apuntan a la pres-
encia de dos sub-poblaciones, delimitadas cada una
por condiciones ambientales locales.

Keywords: WARDMLM.SAS, phytochemical screen- Palabras clave: WARDMLM.SAS, tamizaje fito-

ing, Verbenaceae, intra/interindividual variation químico, Verbenaceae, variación intra/interindividual

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 Revista de Investigación Agraria y Ambiental – Volumen 7 Número 2 – julio - diciembre de 2016 – ISSN 2145-6097
Resumo
A flutuação fisiológica dos constituintes orgânicos ao
redor de sua resposta ambiental têm dois componen-
tes, variação intra e inter individual. Neste estudo, fo-
ram avaliados os componentes da variação biológica
baseado em uma matriz de presença – ausência, ge-
rando valores de precisão inter e intrapopulacional/
individual partindo de uma amostra de 58 acessos
de L. alba. A variabilidade química intraindividual foi
alta (WI=2.06 [98%]) e a interpopulacional, foi baixa
Introduction
Plant essential oils and secondary metabolites.
Essential oils are liquid oily aromatic fragrant [some-
times semi-liquid or solid], obtained from plant ma-
terial [flowers, buds, seeds, leaves, branches, bark,
herbs, Woods, fruits and roots] (UNCTAD, 2005).
Take advantage mainly in the food industry as fla-
voring, the perfume-fragrances industry, and the
pharmaceutical, to add taste and/or smell drugs
(UNCTAD, 2005). Plant secondary metabolites pro-
duction is genetic, performing functions of attrac-
tion-repulsion (Viccini et al., 2004). However, its ex-
pression is determined by pre-existing conditions in
their habitat (presence or absence of phytophagous,
other plant species, biophysical components, other).
These (secondary metabolites), are being actively
used as bioprotectants action _cida [suffix] sustain-
able in current systems of agricultural production in
the world (Das & Das, 2005). The determinants of
chemical variability and yield of essential oil (and its
complex mix of secondary metabolites) in each spe-
cies are important (Figueiredo et al., 2008). These
includes: (a) physiological variations; (b) environ-
mental conditions; (c) geographical variations; (d)
factors genetic and evolutionary; (e) socio-cultural
conditions and (f) quantity of plant material (Ricca-
rdi et al., 2000; Jarvis et al., 2006; Figueiredo et al.,
2008; Palacio & López, 2008).
Composition of the essential oil of Lippia alba.
In L. alba, secondary metabolites are mainly flavo-
noids, produced by epidermal glands in the meso-
phyll cells. Essential oil of L. alba has a relative high
40
(AP=0.033), com FST=0.023 p(rand>data=0.07) e pro-
porção 2%. Não foram observados valores altos de
Estimativos de Variância [Est.Var.] que conduzem a
estabelecer Diversidade Genética-DG- entre popula-
ções e indivíduos. Os resultados gerados pelo teste
apontam á presença de dois subpopulações, delimi-
tadas (cada uma) por condições ambientais locais.
Palavras-chave: WARDMLM.SAS, peneiramento fi-
toquímico, Verbenaceae, variação intra/interindividual
content of carvone, between 30% and 35% (Castro
et al., 2002), turning it into a substitute for other es-
sential oils that contain this same component. An-
other important chemical component of L. alba is
limonene, making up more than 25% of essential oil
(UNCTAD, 2005). The main compounds of the es-
sential oil observed in leaves are: terpinene, p-cy-
mene, caryophylene, myrcene, geraniol, neral and
(Castro et al., 2002.; Jarvis et al., 2006; Blanco et
al., 2007; Celis et al., 2007; Mesa et al., 2009; Esco-
bar et al., 2010).
Chemical variability of essential oils in L. alba.
The chemical composition of the essential oil of L.
alba depends on the geographical origin, the condi-
tions of culture, age and part of the plant used for
extraction, and geobotanical factors (Stashenko et
al., 2003). Genotypic determination (Montanari et
al., 2004; Viccini et al., 2004-2006; Dias et al., 2005-
2006; Suárez et al., 2007; Yamamoto et al., 2008;
Martínez et al., 2008), biochemical characterizations
(Fischer et al., 2004; Hennebelle et al., 2006) and
morphological descriptions (Montanari et al., 2004;
Hennebelle, 2007); they make up the bulk of stud-
ies reported in the species L. alba. Individuals (L.
alba) collected in the northeastern region of Colom-
bia, was shown to be a new chemotype, not previ-
ously described in the literature, containing carvone
[40-57%] as a major component in oils, followed
by limonene [24-37%], biciclosesquifelandreno [2-
22%] piperitenona [1-2%], piperitone [0.8-1.2%] and
the β-burboneno [0.6-1.5%]; the component main in
 Phytochemical variability between Colombian accessions of Lippia alba (Mill.) N.E. Brown
volatile fractions was limonene [27-77%], followed by
carvone [14-30%]], biciclosesquifelandreno [1-33%]
and the β-burboneno [0.5-6.5%] (Stashenko et al.,
2003). The analysis of essential oils obtained from
leaves of samples of French Guiana, Martinique and
Guadeloupe, showed seven different ‘chemotypes’
(Hennebelle et al., 2006), with a possible connection
between chemotype and morphotypes (Hennebelle
et al., 2006). The GxE interaction of 10 genotypes of
four chemical compositions (chemotype) of L. alba
from three regions of the State of São Paulo-Brazil,
had a wide range of leaf production, while essential
oils performance had a high and uniform genotypic
determination. No qualitative variation was detected
for chemical composition and quantitative variation
was of low magnitude. Linalool and limonene geno-
types / carvone were invariably more productive for
performance of oil that the citral genotypes and myr-
cene/canfor (Yamamoto et al., 2008).
Lippia alba (Miller) N.E. Brown is characterized by a
great variability in morphology and chemical compo-
sition of the essential oil (Oliveira et al., 2006). Tava-
res et al. (2005), presented data on the quantitative
variation of the major volatile components of linalol
production in a chemotype of L. alba. The contents
of [alpha] - pinene, (Z) - 3-hexenilo and [alpha] - gur-
junene was higher in the mother plants cultivated
in the ground that in the seedlings grew up in MS
medium, while the contents of sabinene, myrcene,
1.8 - cineole and p-menta - l, 5, 8-trieno was lower.
The addition of 0.2.3 μM of IAA in the middle was
significantly higher for the myrcene content and sa-
binene. The addition of 0.92 μM kinetin increased
significantly 3 (S) - (+) - linalool level (Tavares et al.,
2005). In Colombia, Camargo & López (2008) and
Palacio López (2008) evaluated the response of L.
alba to the availability of water and nitrogen (Antoli-
nez & Rodriguez, 2008). The quantitative variations
of the main volatile components of the production of
linalool in L. alba are associated with nutritional and
environmental factors (Tavares et al., 2005; Camar-
go & López, 2008; Palacio & López, 2008; Antolinez
& Rodriguez, 2008).
Chemical composition and antimicrobial activity of
the essential oils in L. alba were investigated by
Oliveira et al., (2006) to relate them to their tradition-
al uses. The analysis allowed the identification of
two chemotypes of L. alba, a myrcene-citral as che-
motype (15% and 37.1%, respectively) and Lippia
alba f. intermedia as a chemotype citral (22.1%). The
essential oils of both species were active against all
the tested organisms (bacteria and fungi) by halos of
inhibition testing with range from 1.1 to 5.0 cm; prob-
ably due to its high content of oxygenated monoter-
penes (51.0% and 40.1%, respectively), mainly rep-
resented by aldehydes and alcohols. Chemical and
pharmacological data of L. alba obtained by Oliveira
et al., (2006) agreed with the Ethnobotanic survey.
In Colombia, “El Centro de Investigaciones en Bio-
moleculas-CIBIMOL”-from Universidad Industrial
de Santander has made, among others, evaluative
studies on tropical diseases, bioactivity and active
ingredients of Lippia alba how antimicrobial (Bue-
no & Stashenko, 2009); antiviral [Ocazionez et al.
(2010) in the dengue virus; [Meneses et al., (2009)
in the yellow fever virus], antifungal Candida and As-
pergillus (Montiel et al., 2007), in bacteria [Bueno
et al., (2009) in M. tuberculosis]; as anti-Protozoan
(Escobar et al., 2010). A complete list of reports
places it in http://tux.uis.edu.co/quimica/ investiga-
cion/centros/ cibimol.
The goal in the present investigation was to deter-
mine the chemical variability of 58 accessions of L.
alba in such a way to admit an approach to the pop-
ulation structure of the species in Colombia. Been
achieved, through the construction of an array of
chemical data which allowed: a) determine chemical
variability and b) forming a molecular and ecological
data-compatible database that defined the genetic
and spatial structure of the Colombian population of
L. alba.
Materials and methods
Plant material. 200 g of aerial tissue samples
(stems-leaves-flowers mix) of 58 accessions of L.
alba collected in two agro-ecological zones of the
Colombian Andean Region. Accessions formed part
of a transient Bank in vivo ex situ of L. alba of the
“Centro Experimental Universidad Nacional-Sede
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 Revista de Investigación Agraria y Ambiental – Volumen 7 Número 2 – julio - diciembre de 2016 – ISSN 2145-6097
Palmira (CEUNP)”. The study considered two eco-
systems of Bs-T [Chicamocha Region and Suma-
paz Region]. Tropical dry forest corresponds to the
Tropical Alterhidric zonobiom that develops in the
lowlands between 0 and 1000 meters. Detailed in-
formation about zonobiomas on: http://www.hum-
boldt.org.co/es/.
Phytochemical screening (García, 2003; Gar-
cía et al., 2003; García, 2006; Baldizán et al.,
2006) and estimates of variance (Peakall &
Smouse, 2006). Tests [twice] colorimetric with
ethanol and chloroform as solvents, valued the
presence (1)-absence (0) of seven groups of sec-
ondary metabolites in the work sample, encoded
with the Munsell system (tone, value and chroma).
Estimates of variance among and within popula-
tions (Chicamocha and Sumapaz) and individuals
were estimated using the algorithm developed by
Peakall & Smouse (2006). The GenAlex program
(Peakall & Smouse, 2006) applied to the array of
data from laboratory (presence/absence of me-
tabolites) from dual tests with ethanol and chloro-
form as solvents, to assess the presence of seven
secondary metabolites [1. derivatives of couma-
rins, 2. steroids and terpenoid, 3. flavonoids, 4.
cardiac glycosides, 5. saponins, 6. glycosides
and 7. tannin] in 58 Colombian accessions of L.
alba. Tests were developed between December
2012 and January 2013, using the Protocol and
the supervision of the team, from the Phytochem-
istry Laboratory at the Universidad National de
Colombia-Sede Palmira. Bank samples, available
in Lab. Biol. Molec. in UN-Palmira.
Analytical methodologies. Two groups of pro-
cedures were used, Group I (DISTANCE, CLUS-
TER-TREE) and group II (FACTOR, PRINQUAL-
BIPLOT) SAS/STATv9.0; the WARDMLM.SAS
strategy and the GenAlEx program (Peakall &
Smouse, (2006). Step I: raw data were processed
by DISTANCE+ CLUSTER-TREE procedures us-
ing complete linkage distance to find possible
chemical groups. Step II. Laboratory data matrix
was analyzed with procedures PRINQUAL and
SAS/STAT FACTOR v9.0 to obtain an optimal
number of common factors (continuous synthetic
42
variables), the matrix conformative last for the joint
final analysis (ecological, chemical and molecular
data, available in Cardona (2014). A final matrix
approved by common and unique factors was pro-
cessed to determine conclusive analysis and num-
ber of groups. In addition, the WARD MLM.SAS
strategy (supplemented with location data) was
used to identify possible chemical groups, more a
Biplot with SAS/STAT PRINQUAL-FACTOR.
The Analysis of Factors with Probability Maxims-
AFPM, was the research base. The AFPM calcu-
lates estimates of preferential commonality with
values of maximum likelihood with the use of the
PRIORS = SAS/STATV9.0 MAX, in the presence
of singular matrices. The usual form of the analy-
sis is:
proc factor data=SAS-data-set method=principal scree
mineigen=0 priors=max outstat=<libname>;
run;
The square of the multiple correlation (CCM) of
each variable with all other variables was used as
extremely preferential communality [or more impor-
tant] (in: SAS/STATv9.0); and it was basic in the final
formation of groups.
Interpretation of analysis. The array generated
by the test data were analyzed using the algorithm
developed by the program GenAlex v.6.5 (Peakall &
Smouse, 2006) for binary data (1/0). The data matrix
was formed by 58 accessions (35 of the Region I =
Sumapaz and 23 Region II = Chicamocha). The re-
sults of the analysis of variance are obtained from a
distance matrix to calculate the statistical F based on
the formulas:
Fst = AP / (WI + AI + AP) = AP / TOT
Fis = AI / (WI + AI)
Fit = (AI + AP) / (WI + AI + AP) = (AI + AP) / TOT.
Inbreeding coefficient
Nm = [(1 / Fst) - 1] / 4 number effective
migrants per generation
 Phytochemical variability between Colombian accessions of Lippia alba (Mill.) N.E. Brown

Results given by the ANOVA [MS = 2.567]. The variability

The analysis of variances (from phytochemicals
data) shown in Table 1. Chemical variability within
individual [WI = 2 060] had high and between popu-
lations [AP = 0. 033], indicator low. Among individu-
als, the analysis showed zero [AI = 0]. AP was the
source of variation with more weight on the answer
between populations [AP = 0. 033] was low with a
FST = 0.023 P(rand≥data=0.07) and 2% proportion.
However, the estimate of variance with the largest
proportion occurred within individuals WI = 98%.
The greater variability occurred within individual.
The maximum and minimum values for FST were
0.597 and 0.038, respectively.

Table 1. Estimates of variance [Est.Var.] for 58 Colombian accessions

of L. alba phytochemical screening, using program GenAlex v6.5.

Source Df SS MS Est. Var. Proporción

Among Pops(AP) 1 2.567 2.567 0.033 2%

Among Indiv(AI) 56 42.493 0.759 0.000 0%

Within Indiv(WI) 58 119.500 2.060 2.060 98%

Total 115 164.560 2.093 100%

F-Statistics Value P (rand >= data)

Fst 0.023 0.070

Fis -0.462 1.000

Fit -0.429 1.000

Fst máx. 0.597

F’st 0.038

Nm 10.819

Main discussion. The degree of differentiation phy-
tochemical (by presence-absence of secondary me-
tabolites) between populations of 0.03, indicates a
similarity between the two populations, ‘probably’
because of their form of dissemination of the species
in Colombia (Anthropochory). However, this 2% indi-
cates a minimum portion of individuals with maximum
degree of differentiation caused by allogamy and low
viability of seed; demonstrating sexual reproduction
and differentiation among populations most likely be-
cause of allogamy, more local environmental factors.
Estimates of variance between regions indicate similar
populations and among individuals not differentiable
chemotypes it shows. The estimate of variance within
individual indicated high variability (98%) for second-
ary metabolites-sm-caused by physiological and envi-
ronmental response content. The degree of differentia-
tion for presence/absence of sm within and between
individuals, had negative values (Table 1), indicating
that individuals are chemically similar probably due to
their low capacity of sexual reproduction and its form
of propagation-dissemination (Anthropochory=active
dissemination of reproductive material, due to human
action). The possible chemical groups are displayed
in the dendrogram of Figure 1. The range of values of
F obtained our possibility: a) of identifying at least one
special and b) discriminating chemotypes of the sm in
the final formation of groups.

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 Revista de Investigación Agraria y Ambiental – Volumen 7 Número 2 – julio - diciembre de 2016 – ISSN 2145-6097

Number chemical groups present in the work- and E=0.6271) and Flavonoids (C=0.6271 and
ing sample. The WARDMLM.SAS strategy deter- E=0.7458); and low for glycosides (C=0.0169
mined the presence of nine phytochemicals groups and E=0) and cardiac glycosides [C=0.0508 and
(Table 2). The final number of groups, shows nine E=0.0508]. Derivatives of coumarins and saponins
phytochemical groups: Group I (accessions 189- showed intermediate values. Glycosides and het-
359-360-362/3/4/5); Group II (174-201/2-209- erosides were exceptionally low, 0.05 and 0.01 re-
373/4/5/6/7/8); Group III (300/1/2/3/4/5); Group IV spectively (Table 3).
(346-348/9-350/1/2/3/4/356/7/9); Group V (CEUN-
308-310/1/2/3/4-320-322); Group VI (3-4-5); Group Table 3. Proportion of individuals with value [1] pres-
ence of seven types of secondary metabolite
VII (369-370); Group VIII (187-366/7); Group IX in two types of solvent.
(340/1/2/3/4/5-347). However, the Biplot of Figure
2 suggests a predominant majority chemical group Metabolites Solvent Frecuency
[in Colombia] and the presence of at least three ac-
Cloroformo 0.1186
cessions with individual chemical characteristics Derivates of Cumarins
by the presence-absence of sm in 201, 213, and Etanol 0.1186
341 accessions. Its location within the Biplot indi-
Cloroformo 0.8305
cated chemical properties own and different from Esteroids & Terpenoids
the rest of accessions included in the test. This re- Etanol 0.6271
sult suggests the ‘possible’ collection of individuals
Cloroformo 0.6271
(sexually) propagated by seed. Group V, included Flavonoids
the CEUN accession of UN-Palmira. Etanol 0.7458

Table 2. Final number of groups and individuals by Cloroformo 0.0508

Group [weight-Proportion] obtained with the Glycosides inotropic agent
WARDMLM.SAS strategy. Etanol 0.0508

Cloroformo 0.3729
Group Frecuency Weight Proportion
Saponins
1 7 7.0000 0.118644 Etanol 0.1356

2 10 10.0000 0.169492 Cloroformo 0.0169

Heterosides Cianogenics
3 6 6.0000 0.101695 Etanol 0.0000

4 11 11.0000 0.186441 Cloroformo 0.8136

Tanins
5 9 9.0000 0.152442 Etanol 0.6949

6 4 4.0000 0.067797

7 3 3.0000 0.050847
Discussion
8 3 3.0000 0.050847

9 6 6.0000 0.101695 The phytochemical screening showed the absence

The outstanding frequencies were, in order: Ste-
roids & Terpenoids > Tannins> Flavonoids, the
lower frequency was in glycosides and cardiac gly-
cosides. The results of Table 3 show high frequen-
cies to Tannins (chloroform-[C] = 0.8136 and etha-
nol [E] = 0.6949), Steroids & Terpenoids (C=0.8305
of alkaloids in Lippia alba (Morataya, 2006; Medina-
Lopez et al., 2011). With nulls to alkaloids, WARD-
MLM.SAS strategy defined a final number of nine
phytochemicals groups. For the number of groups
technical support defined it strategy in step III, gen-
erating a list of the log-Likelihood corresponding to
each of the possible numbers of groups and the
graphic logL versus number of groups.

44
 Phytochemical variability between Colombian accessions of Lippia alba (Mill.) N.E. Brown
High frequency of steroids & amp; Terpenoides and
flavonoids stock defines the chemotypes present in
Colombia. Some medicinal properties of Lippia alba
are due to the high content of lightweight terpenes
as 1, 1,8-cineole, limonene, b-myrcene (Riccardi et
al., 2000). The antioxidant activity and/or antimicro-
bial activity are attributed to flavonoids and phenols/
tannins (Martínez et al., 2002; Núñez et al., 2008)
Phytochemicals lower frequencies reveal the exis-
tence of other possible chemotypes for Colombia.
Contrasting values of frequency indicate the pres-
ence of a dominant majority group in Colombia.
The WARDMLM.SAS strategy shows three major-
ity groups being more discriminating than the Biplot.
The results obtained by the different tests suggest
that variability Phytochemistry of the sample of L.
alba, is a physiological-environmental and there
are no involved epigenetic factors. Analysis genetic
markers RAM, confirm it (Cardona, 2014).
Analysis of factors with maximum likelihood.
The cluster with the Ward method and the distance
for Gower, formed two groups, one consisting of
the 187-356-340-454-370-367-369-341 accessions
Figura 1. Phytochemical groups based in SAS/STATv9.0 AFPM for net laboratory data.
and the second large group composed of three
subgroups (Figure 2). Accession 341 were com-
pletely independent. The cluster shows that they
are chemically equal each other: the accessions (1-
351); (356-359-360-378); (362/3/4/5-374/5); (4-320);
(174-189-373); (202-209-217-300-302/3/4/5-377);
(301-346-344). The Biplot generated by factors of
commonality-FC is shown in Figure 2.
AFPM (analysis of factors with high probabil-
ity) procedure used the option PRIORS = MAX at
presence of unique correlation matrices. The ei-
genvalues of the reduced matrix of correlations
(Total=10.2698937, average=0.51349468) defined
11 factors with the resembles criteria explaining the
1.1464 variation found in the test. The matrix of 11
factors including region, altitude and coordinates
(North and West) decimal places, is part of the fi-
nal matrix of joint markers that defined the genetic
structure and phytogeographic samples of Lippia
alba used in the test. This output generated by the
AFPM matrix markers phytochemicals that are gen-
erated from the respective Biplot and phytochemical
cluster is considered. The Biplot and cluster gener-
ated by the AFPM are shown in Figures 1 and 2.
45
 Revista de Investigación Agraria y Ambiental – Volumen 7 Número 2 – julio - diciembre de 2016 – ISSN 2145-6097

Figure 2. Biplot for factors with values of maximum-likelihood [FPM] presence of ms 58

Colombian accessions of Lippia alba, including data from site [region, altitude, coordinates].

Discussion. The Biplot and cluster shows four major
trends, separating chemotypes into two large groups:
Region Chicamocha and Sumapaz region. There are
homologies within region and possible division into
two sub-populations, determined by environmental
conditions. Taking into account region, altitude, and
coordinate strategy wardmlm.sas determined for the
Region Chicamocha, four groups and to the Suma-
paz Region five groups. The cluster shows the vari-
ability (WI) within and between sub-populations. The
two sub-populations were determined based on loca-
tion data and the values of Est.Var. obtained by the
ANOVA. The environmental factor that predominates
in Lippia alba and has been supported by several au-
thors including Stashenko et al. (2003). the net chem-
ical variability, is fully defined in Table 1.
Conclusions
There were no high values of Estimates of vari-
ance [Est.Var.] leading to establish chemical vari-
ability among individuals and populations. The re-
sults generated by the test point to the presence
of two subpopulations, defined (each) by local en-
vironmental conditions.

46
 Phytochemical variability between Colombian accessions of Lippia alba (Mill.) N.E. Brown
The high value of WI indicated the possible pres-
ence of individuals with important chemical vari-
ants. The Verbenaceae tend to have chemical
variations, particularly Lippia alba, without show-
ing morphological differences among provenances
(Riccardi et al., 2000). These variations occur even
in plants located at short distances (Riccardi et al.,
2000). 201, 213, and 341 accessions are variants
important phytochemicals.
Biological variability can be original epigenetic, ge-
netic, environmental, both, in response to Herbiv-
ory or mechanical damage and latrogenic (by pol-
lution, especially chemical agents). Assuming, that
the Individual variability [WI] must be less than the
variability between individuals [AI], [WI] individual
biological variability and maximum biological vari-
ability to estimate total [WT], allowed get popula-
tion reference values and set values of variability
for working sample.
The variables of location [including the environ-
mental] explain the phytochemical variability of Lip-
pia alba in large proportion. The working sample
reported very wide range of adaptability, from 0 in
Buenaventura-Valle, up to 2800 meters altitude in
the Department of Cauca; revealed by the value of
Phytochemical high intra variability (of 98%) found in
the test (WI = 2.06).
The phytochemical characteristics of the sub-
populations correspond to those reported by Co-
lombian authors, with important chemical vari-
ants and value of extremely of variance [Est. Var.]
also important. The examination of essential oils
showed components lightweight terpenes as ma-
jority, alongside tannins and flavonoids. Thus, a)
the sample of work corresponds to the chemotypes
already documented by other authors for Colombia,
and most likely others not yet reported, validating
the laboratory data obtained; and (b) the similarity
observed in the sample determines the accessions
phytochemical kinship, which allows the formation
of a large chemical group dominated by tannins-
Terpenoids-flavonoids.
Cited literature
1. Antolinez-Delgado, C. A. & Rodríguez-López, N. (2008).
Plasticidad fenotípica en Lippia alba y Lippia origan-
oides (verbenaceae): respuesta a la disponibilidad de
nitrógeno. Acta biol. Colomb., Vol. 13 No. 1, 53 – 64.
2. Baldizán, A., Domínguez, C., García, D. E., Chacón, E.
& Aguilar, L. (2006). Metabolitos secundarios y patrón
de selección de dietas en el bosque deciduo tropical
de los llanos centrales venezolanos. Rev Zoot Trop, Vol
24, No 3, pp 213-232.
3. Blanco KM, A.J. Agudelo, J.R. Martínez, E. & Stash-
enko, E.. (2007). Estudio comparativo de los aceites
esenciales de Lippia alba Mill N.E. Brown cultivada
con tres tipos de compostaje, Scientia et Technica,
año XIII, 33, 231-233.
4. Bueno J.G. & Stashenko, E. (2009). Antimycobacterial
natural products an opportunity for the Colombia biodi-
versity, Revista Española de Quimioterapia, Sometido.
5. Bueno, J. G., Martínez, J. R., Stashenko, E. & Ribón,
W. (2009). Anti-tubercular activity of some aromatic
and medicinal plants, grown in Colombia, Biomédica,
2009, 29 (1), 51-60.
6. Camargo, A. A. & Rodríguez López, N. (2008). Expresión
fenotípica de la asignación de biomasa en Lippia origan-
oides y Lippia alba: Repuestas a la disponibilidad de agua
en el suelo, Acta Biológica Colombiana, 13 (3), 133-148.
7. Cardona, J. O. (2014). Estructura genética y fitogeografía
de poblaciones colombianas de Lippia alba (Mill) N.E.
Brown (Verbenaceae). (Tesis de Doctorado en Ciencias
Agrarias Línea de investigación Mejoramiento Genético
Vegetal). Universidad Nacional de Colombia-Sede Palmira
Facultad de Ciencias Agropecuarias. Palmira, Colombia.
8. Celis, C. N., Escobar, P., Isaza Martínez, J. H., Stash-
enko, E. & Martínez, J. R. (2007). Estudio comparativo
de la composición química y la actividad biológica de
los aceites esenciales de Lippia alba, Lippia origan-
oides y Phyla dulcis, especies de la familia Verbena-
ceae, Scientia et Technica, año XIII, 33,103-105.
9. Castro, D.M., Ming, L.C. & Marques, M. O. M. (2002).
Biomass production and chemical composition of Lip-
pia alba (MILL.) N.E.BR. ex britt & wilson in leaves on
different plant parts in different seasons. Acta Hort.
(ISHS) 569:111-115. Recuperado de: http://www.acta-
hort.org/books/569/569_18.htm
10. Das, T., & Das, A. K. (2005). Inventorying plant biodiver-
sity in homegardens: a case study in Barack Valley, As-
sam, North East India. Current Science 89, 1, 155-163.
11. Escobar, P., Leal, S. M., Herrera, L. V., Martínez, J. R.
& Stashenko, E. (2010). Chemical composition and an-
tiprotozoal activities of Colombian Lippia spp essential
oils and their major components. Mem. Inst. Oswaldo
Cruz, 2010, 105, 184-190.
12. Figueiredo, A. C., Barroso, J.G., Pedro, L. G. & Schef-
fer, J. J. C. (2008). Factors affecting secondary metabo-
lite production in plants: volatile components and es-
sential oils. Flavour and Fragrance Journal, Volume 23
Issue 4, Pages 213–226.
47
 Revista de Investigación Agraria y Ambiental – Volumen 7 Número 2 – julio - diciembre de 2016 – ISSN 2145-6097
13. Fischer, U., Lopez, R., Poell, E., Vetter, S., Novak, J.
& Franz, C.M. (2004). Two chemotypes within Lippia
alba populations in Guatemala. Flavour and Fragrance
Journal 19, 333-5. Contact: Novak, Johannes; Inst Appl
Bot, Univ Vet Med, Vet Pl 1, A-1210, Vienna, Austria.
14. García, D. E. (s. f.). Evaluación de los principales fac-
tores que influyen en la composición fitoquímica de
Morus alba (Linn.). Tesis de Maestría, Estación experi-
mental de Pastos y Forrajes “Indio Hatuey”, Cuba. 97 pp.
15. García, D. E., Medina, M. G., Domínguez, C., Baldizán,
A., Humbría, V. & Cova, L. (2006). Evaluación química de
especies de leguminosas con potencial forrajero en el Es-
tado de Tujillo, Venezuela. Rev Zoot Trop, 24 (4): 401-415.
16. Hennebelle, T., Sahpaz, S., Dermont, C., Joseph, H. &
Bailleul F. (2006). The essential oil of Lippia alba: analysis
of samples from French overseas departments and review
of previous works. Chem Biodivers. Oct; 3(10): 1116-25.
17. Hennebelle, T., Sahpaz, S., Gressier, B., Joseph, H. &
Bailleul, F. (2007). Antioxidant and neurosedative prop-
erties of polyphenols and iridoids from Lippia alba.
Phytother Res. Aug 17: 17705148 (P,S,E,B,D).
18. Hennebelle, T., Sahpaz, S., Joseph, H. & Bailleul, F.
(2008). Ethnopharmacology of Lippia alba. France. J.
Ethnopharmacology, Mar 5; 116(2): 211-222.
19. Jarvis, A., Paternina, M. J., Arcos, A., Rodríguez, H. J.,
Nagles, C. & Melo, N. (2006). Evaluación Rápida de la
Adaptación al Medioambiente de plantas promisorias
medicinales. En: II Congreso internacional de plantas
medicinales y aromáticas. Universidad Nacional de
Colombia-Sede Palmira, Octubre 19-20 y 21.
20. Martínez, F. (2008). “Diversidad y relaciones genéticas
de plantas promisorias de jardín del género Lippia en
el Departamento de Santander, Colombia”. GIEFIVET-
UIS. Producción científica. Proyectos de investigación
finalizados. Recuperado de: http://cenivam. uis.edu.co
/cenivam/productos/proinvesfinalizados.html.
21. Medina-López, L. A., Araya-Barrantes, J. J., Tamayo-
Castillo, G. & Romero, R. M (2011). Comparación de
metodologías de extracción para limoneno y carvona
en Lippia alba usando cromatografía de gases. Cien-
cia y Tecnología, 27(1y 2): 1-13,2011. ISSN: 0378-0524.
22. Meneses, R., Ocazionez, R., Martínez, J.R. & Stash-
enko, E. (2009). Inhibitory effect of essential oils ob-
tained from plants grown in Colombia on yellow fever
virus replication in vitro. Annals of Clinical Microbiol-
ogy and Antimicrobials, 8 (8), Recuperado de: http://
www.ann-clinmicrob.com/ content/8/1/8.
23. Mesa Arango, A. C., Montiel Ramos, J., Zapata, B.,
Durán García, D.C., Betancur Galvis, L. & Stashenko,
E. (2009). Citral and carvone chemotypes from the es-
sential oils of Colombian Lippia alba (Mill.) N.E. Brown:
composition, cytotoxicity and antifungal activity, Memo-
rias do Instituto Oswaldo Cruz, 2009, 104 (6), 878-884.
24. Montanari, R.M, Sousa, L. A., Leite, M.N., Coelho,
A.D.F., Viccini, L.F. & Stefanini, M.B. (2004). Phenotypi-
cal plasticity of the external morphology in Lippia alba
(Mill.) N. E. Br. ex Britt & Wilson in response to level of
luminosity and fertilization. Revista Brasileira de Plan-
tas Medicinais (6), 96-101.
48
25. Montiel, J., Mesa Arango, A.C., Durán García, D.C.,
Bueno, J. G., Betancur Galvis, L. A. & Stashenko, E.
2007. Evaluación de la actividad anti-candida y an-
ti-aspergillus de aceites esenciales de Lippia alba
(Miller) N.E Brown y su asociación con sus compo-
nentes mayoritarios Scientia et Technica, año XIII,
33, 243-246.
26. Morataya-Morales, M. A. (2006). Caracterización
farmacopéica de cuatro plantas nativas de Guatemala
Albahaca (Ocimum micranton), Orégano (Lippia gra-
veolens), Salvia sija (Lippia alba) y Salviyá (Lippia
chiapaensis). (Tesis para optar al título de Química
Farmaceútica). Universidad de San Carlos de Guate-
mala, Facultad de Ciencias Químicas y Farmacia.
27. Núñez María, B., Sánchez Edit, G., Bela, A. & Aguado,
M. I. (2008). Determinación de metabolitos secundar-
ios en Lippia alba (Mill.) y Lippia turbinata (Griseb.)
Universidad Nacional del Nordeste. Secretaria Gen-
eral de Ciencia y Tecnología. Comunicaciones Cientí-
ficas y Técnicas.
28. Ocazionez, R. E., Meneses, R., Torres, F. A. & Stash-
enko, E. (2010). Virucidal activity of Colombian Lippia
essential oils on dengue virus replication in vitro. Mem.
Inst. Oswaldo Cruz, 105, 304-309.
29. Oliveira, D. R., Gilda, L., Santos, S., Bizzo, H., Lopes,
D., Alviano, C. & Leitão, S. (2006). Ethnopharmacologi-
cal study of two Lippia species from Oriximiná, Brazil.
Journal of Ethnopharmacology Volume 108, Issue 1, 3
November, Pages 103-108.
30. Palacio, K. & Rodríguez López, N. (2008). Plasticidad
fenotípica de dos poblaciones de Lippia alba (Ver-
benaceae) sometidas a déficit hídrico bajo dos condi-
ciones lumínicas, Acta Biológica Colombiana, 13 (1),
187-198.
31. Peakall, R. & Smouse, P. E. (2012). GenAlEx6.5: ge-
netic analysis in Excel. Population genetic software for
teaching and research-an update. Bioinformatics Ap-
plication Note. Vol 28 no. 19 2012, pages 2537-2539.
doi:10.1093/bioinformatics/bts460.
32. Peixoto, P., Salimena, F. & Santos, M. D. (2006) In vi-
tro propagation of endangered Lippia filifolia Mart.
and Schauer ex Schauer. In Vitro Cell Dev Biol Plant
42:558–561.
33. Riccardi, G., Ricardi, A. & Bandoni, A. (2000). Fito-
química de Verbenaceae (Lippias y Aloysias) del Nor-
deste argentino. Universidad Nacional del Nordeste.
Comunicaciones Científicas y Técnicas.
34. Stashenko, E.E., Jaramillo, B.E. & Martínez, J. R.
(2003). Comparación de la composición química y de
la actividad antioxidante in vitro de los metabolitos se-
cundarios volátiles de plantas de la familia Verbena-
ceae. Rev. Acad. Colomb. Cienc. 27 (105): 579-597.
ISSN 0370-3908.
35. Stashenko, E. E., Jaramillo, B. E. & Martínez, J. R.
(2004). Comparison of different extraction methods for
the analysis of volatile secondary metabolites of Lippia
alba (Mill.) N.E. Brown, grown in Colombia, and evalu-
ation of its in vitro antioxidant activity, J. Chromatogr. A,
1025, 93-103
 Phytochemical variability between Colombian accessions of Lippia alba (Mill.) N.E. Brown
36. Suárez, A.R., Martínez, F. O., Castillo, G. A. & Chacón,
M . I. (2007). Molecular Characterization of Aromatic
Species of the Genus Lippia from the Colombian Neo-
tropics. Proceedings of the International Symposium
on Medicinal Nutraceutical Plants. Ed. A.K. Yadak. Acta
Hort. 756, ISHS
37. Tavares, E. S., Juliao, L. S., Lopez, D., Bizzo, H. R.,
Lage, C. L. S. & Leiao, S. G. (2005). Análise do óleo
escencial de folhas de três quimiotipos de Lippia alba
(Mill.) N.E. Br. (Verbenaceae) cultivados em condições
semelhantes. Revista Brasilera de Farmacognosia
15(1):1-5,Jan./Mar.
38. UNCTAD. (2005). Market Brief in the European Union
for selected natural ingredients derived from native
species, Lippia alba (Prontoalivio, Erva cidreira, jua-
nilama, Melissa). This publication was developed in
the context of the UNCTAD / BioTrade Facilitation. Pro-
gramme, funded by the Governments of the Nether-
lands and Switzerland.
39. Viccini, L. F., Souza da Costa, D. C., Machado, M.
A. & Campos, A. L. (2004). Genetic diversity among
nine species of Lippia (Verbenaceae) based on RAPD
Markers. Plant Systematics and Evolution. Volume
246, Numbers 1-2
40. Viccini, L. F., Pierre, P. M. O., Praça, M. M., Souza da
Costa, D C., da Costa Romanel, E., de Sousa, S.M.,
Peixoto, P. H., Salimena, P. & Gonçalves, F. R. (2006).
hromosome numbers in the genus Lippia (Verbenace-
ae). Revista Plant Systematics and Evolution; 256 (1-4)
Página(s):171-178 ISSN: 03782697.
41. Yamamoto, P., Colombo, C. A., Azevedo Filho, J. A.,
Lourencao, A. L., Mayo Marques, M., da Silva Mo-
rais, G., Chiorato, A. F., Melo Martins, A. L. & Siquei-
ra, W. J. (2008). Perfomance of ginger grass (Lippia
alba) for traits related to the production of essential
oil. Sci. Agric. (Piracicaba, Braz.), v.65, n.5, p.481-
489, Sept. /Oct.
Conflicto de Intereses
Los autores declaran no tener
ningún conflicto de intereses
Recibido: 03 de marzo de 2016
Aceptado: 21 de abril de 2016
49